Design, synthesis and methods of use of acyclic fleximer nucleoside analogues having anti-coronavirus activity

ABSTRACT

The present invention is directed to compounds, methods and compositions for treating or preventing viral infections using nucleosides analogs. Specifically, the present invention provides for the design and synthesis of acyclic fleximer nucleoside analogues having increased flexibility and ability to alter their conformation structures to provide increased antiviral activity potential with the result of inhibiting several coronaviruses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/546,818, filed on Jul. 27, 2017, Now U.S. Pat. No. 10/058,516 whichwas filed under the provisions of 35 U.S.C. § 371 and claims thepriority of International Patent Application No. PCT/US2016/015327 filedon Jan. 28, 2016 which in turn claims priority to U.S. ProvisionalApplication No.: 62/109,667 filed on Jan. 30, 2015 and U.S. ProvisionalApplication No.: 62/195,968 filed on Jul. 23, 2015, the contents of bothare incorporated by reference herein for all purposes.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant NumberR21AI097685 and T32GM066706 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF INVENTION

The present invention is directed to compounds, methods and compositionsfor treating or preventing viral infections using nucleosides analogues.Specifically, the present invention provides for the design andsynthesis of acyclic fleximer nucleoside analogues having increasedflexibility and ability to alter their conformation to provide increasedantiviral activity potential with the result of inhibiting severalcoronaviruses.

BACKGROUND OF THE INVENTION

Nucleoside analogues as a class have a well-established regulatoryhistory, with many currently approved by the US Food and DrugAdministration (US FDA) for treating viruses and cancer, including butnot limited to leukemias, lymphoms, cervical cancer, skin cancers, humanimmunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus(HCV), herpes simplex, varicella zoster virus (VZV) and respiratorysyncytial virus (RSV), among others. However, there is a currentchallenge in developing cancer and antiviral therapies to inhibit cancercells or viral replication without injuring the host cell.

Currently there are no approved treatments or vaccines for humancoronaviruses (HCoVs) or potentially lethal zoonotic coronaviruses(CoVs), such as severe acute respiratory syndrome (SARS) or Middle Eastrespiratory syndrome (MERS). HCoVs were first identified in the 1960swith only two species known at the time, HCoV-229E and HCoV-OC43. Theseviruses are known to cause a large number of common colds with typicallymild symptoms, with the exception of those suffering from otherillnesses, particularly immunocompromised systems. (9) In 2002 a newcoronavirus pathogen associated with severe lung disease emerged inGuangzhou, and later spread to Southern China and Hong Kong. The newvirus was named SARS-CoV, (10) and before the end of the outbreak over8,000 cases were confirmed in several countries and with almost 8,000fatalities. Since then two additional coronaviruses, HCoV-NL63 andHCoV-HKU1, were discovered in humans and most recently, in 2012,MERS-CoV was identified as a second zoonotic coronavirus that can causelethal respiratory infections in humans.

The current MERS outbreak has been ongoing for almost three years, withwell over a thousand confirmed cases having been documented, with amortality rate of about 40%. (1) Since the 2002-2003 SARS outbreaks,there have been extensive efforts to target the coronavirus family,including the screening of libraries of already approved antiviral drugssuch as acyclovir (ACV), ganciclovir, lamivudine, and zidovudine.Unfortunately, none of these well-known antiviral drugs exhibited anyactivity against SARS-CoV or MERS-CoV in vitro. (2)

The SARS-CoV screening efforts did, however, yield a small number ofleads including the nucleoside analogue ribavirin, a guanosine-likeanalogue that has exhibited broad-spectrum antiviral activity. (2-7)Ribavirin was found to inhibit coronavirus replication in vitro, butwith an inhibitory concentration much higher (500-5000 μg/ml) than thatneeded to inhibit other viruses (50-100 μg/ml). Consequently, it doesnot appear to represent a viable treatment option. Moreover, a recentstudy has suggested that in the case of the coronaviruses, ribavirin'santiviral activity is not primarily due to lethal mutagenesis, butrather to its effect on the cell's Guanosine-5′-triphosphate (GTP)biosynthesis. (8) Beyond these studies, there are few reports ofnucleoside inhibitors being studied or developed to combat coronavirusinfection.

The need for new and more effective antiviral therapeutics, particularlythose targeting emerging and reemerging infectious diseases andpathogens continues to increase. Thus, in light of the above discussion,there is a need for discovering and providing new and more efficientantiviral drugs.

SUMMARY OF THE INVENTION

The present invention provides for flexible and modified nucleosideanalogues that allow access to more potential binding sites with theability to retain their potency against resistant cancers and viralstrains since they can “wiggle and jiggle” in the binding site. Thesefindings are causing a paradigm shift in drug design having anticancerand antiviral activity.

In one aspect, the present invention provides for a series of doublyflexible nucleoside analogues based on the acyclic nucleosides and theflex-base moiety found in the fleximers selected from compoundsaccording to the following:

or pharmaceutically acceptable salt, isomer, hydrate, prodrug or solvatethereof.

In another aspect the present invention provides for nucleosideanalogues based on the acyclic nucleoside acyclovir (ACV) selected fromthe following compounds:

or a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.

More specifically, a nucleoside analogue based on the acyclic nucleosideacyclovir (ACV) is selected from the following compounds the ACV

wherein Ac is CH₃(C═O),or a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.

In yet another aspect, the present invention provides for a nucleosideanalogue based on the acyclic nucleoside acyclovir selected from thefollowing compounds:

or a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.

In another aspect, the present invention provides for the use ofmodified nucleosides of the present invention for medicine. In a morespecific embodiment hereof, said use as a medicine is for the preventionor treatment of a coronavirus, SARS and MERS-CoV, more specifically forthe prevention or treatment of an infection of a coronavirus, SARSand/or MERS-CoV in a subject, mammal or human.

In yet another aspect, the present invention provides for a method oftreating, reducing or preventing the effects of a coronavirus in asubject in need of such therapy, the method comprising administering atherapeutically effective amount of a compound selected from the groupconsisting of

or a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.

More specifically, the method of treating, reducing or preventing theeffects of a coronavirus in a subject in need of such therapy includesadministering a therapeutically effective amount of a nucleosideanalogue based on the acyclic nucleoside acyclovir (ACV) selected fromthe following compounds:

wherein Ac is CH₃(C═O),or a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof; and still more specifically the method of treating,reducing or preventing the effects of a coronavirus in a subject in needof such therapy comprises administering a therapeutically effectiveamount of a compound selected from the group consisting of:

or a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.

Preferably, a therapeutically effective amount of the acyclic fleximernucleoside analogue is from 0.05 to 50 mg per kilogram body weight ofthe subject per day.

In a still further aspect, the present invention provides for a methodof binding to both natural and mutated polymerases of a coronavirus, themethod comprising administering a therapeutic amount of a modifiednucleoside inhibitor, wherein the modified nucleoside is selected fromthe group consisting of:

or a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof;and more specifically the method of binding to both natural and mutatedpolymerases of a coronavirus, comprises a compound selected from thegroup consisting of:

and a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.

In yet another aspect, the present invention provides for contacting acell infected with a coronavirus or to be infected with a coronaviruswith at least one of the modified nucleosides provided herein, whereinthe amount of the modified nucleosides is from about 1 μg/ml to about 40μg/ml, and more preferably, from about 3 μg/ml to about 20 μg/ml.

In another aspect, the present invention provides for the manufacture ofa medicament comprising the modified nucleosides of the presentinvention for the treatment of a coronavirus, SARS and MERS-CoV.

In another aspect, the present invention provides for the use of themodified nucleosides of the present invention for the prevention ortreatment of a coronavirus, SARS and MERS-CoV, more specifically for theprevention or treatment of an infection of a coronavirus, SARS and/orMERS-CoV in a subject, mammal or human.

In yet another aspect, the present invention provides for apharmaceutical composition comprising at least one of the modifiednucleosides of the present invention and a pharmaceutically acceptablecarrier.

In a still further aspect, the present application provides for a methodof treating CoV in a patient, comprising administering to said patient atherapeutically effective amount of a compound of the present invention,and at least one additional therapeutic agent having anti-CoVproperties.

In another aspect, the invention also provides a method of inhibitingCoV, comprising administering to a mammal infected with CoV a compoundselected from compounds 2 and 19 and pharmaceutically effective saltsthereof in an amount to effectively inhibit the replication of CoV ininfected cells in the mammal.

In yet another aspect, the invention also provides novel intermediatesor prodrugs which are useful for preparing the compounds of theinvention or converted to active agents in vivo, respectively. Prodrugsare selected and prepared in order to improve some selected property ofthe molecule, such as water solubility or ability to cross a membrane,temporarily. Most common (biologically labile) functional groupsutilized in prodrug design include carbonates, esters, amino acylesters, amides, carbamates, oximes, imines, ethers or phosphates.

In other aspects, novel methods for synthesis, analysis, separation,isolation, purification, characterization, and testing of the compoundsof this invention are provided.

DETAILED DESCRIPTION OF THE INVENTION

In designing the target molecules the flex-base modification of thefleximers was combined with the acyclic sugar moiety of acyclovir (ACV).ACV is a nucleoside polymerase inhibitor currently approved for thetreatment of herpes simplex virus (HSV) and varicella zoster virus (VZV)infections.(18) Recently, it was also found to have activity againsthuman immunodeficiency virus (HIV) when McGuigan's ProTide technologywas employed.(19) It was found to suppress the replication of both HIV-1and HSV-2 in the submicromolar range in lymphoid and cervicovaginalhuman tissues and at 3-12 μmol/L in CD4⁺ T cells. (19)

Unique nucleoside analogues have been termed ‘fleximers’ and weredesigned to explore how nucleobase flexibility affects the recognition,binding, and activity of nucleoside(tide) analogues. (11-16) Thefleximers possess a purine base scaffold in which the imidazole andpyrimidine moieties are attached by a single carbon-carbon bond, ratherthan being ‘fused’ as is typical for the purines. These analogues aredesigned to retain all of the requisite purine hydrogen bonding patternswhile allowing the nucleobase to explore alternative binding modes.Previous work from the present inventors include flexible analogues haveseveral strategic advantages, such as increased binding affinitycompared to the corresponding rigid inhibitors, binding affinity toatypical enzymes, as well as the ability to overcome point mutations inbiologically significant binding sites.(11, 12, 17)

The present invention provides for a series of doubly flexiblenucleoside analogues based on the acyclic sugar scaffold of acyclovirand the flex-base moiety found in the fleximers. The target compoundswere evaluated for their antiviral potential and found to inhibitseveral coronaviruses. Significantly, several of the compounds displayedselective antiviral activity (CC50>3×EC50) towards human coronavirus(HCoV)-NL63, Middle East respiratory syndrome-coronavirus (MERS-CoV) andsevere acute respiratory syndrome-coronavirus (SARS-CoV). In the case ofHCoV-NL63 the activity was highly promising with an EC50<10 μM and aCC50>100 μM. As such, these doubly flexible nucleoside analoguesdescribed herein are viewed as a novel new class of drug candidates forpotent inhibition of coronaviruses.

Mammal or human hosts infected with a coronavirus can be treated byadministering to said mammal or human an effective amount of an acyclicfleximer nucleoside analogue of the present invention and such compoundscan be administered by any appropriate route, for example, orally,parenterally, intravenously, intradermally, subcutaneously, ortopically, in liquid or solid form.

The present invention relates to a method for treating a CoV viralinfection, comprising the administration, to a patient, of an effectiveamount of at least one acyclic fleximer nucleoside analogue of thepresent invention and/or of a composition containing same. In general,the acyclic fleximer nucleoside analogues, as active agents, of thisinvention will be administered in a therapeutically effective amount byany of the accepted modes of administration for agents that servesimilar utilities. The effective amount will be that amount of anacyclic fleximer nucleoside analogue of the present invention that wouldbe understood by one skilled in the art to provide therapeutic benefits.The active agent can be administered once a week, twice or more timesper week, once a day, or more than once a day. As indicated above, allof the factors to be considered in determining the effective amount willbe well within the skill of the attending clinician or other health careprofessional.

For example, therapeutically effective amounts of an acyclic fleximernucleoside analogue of the present invention may range fromapproximately 0.05 to 50 mg per kilogram body weight of the subject perday; preferably about 0.1-25 mg/kg/day, more preferably from about 0.5to 10 mg/kg/day. Thus, for administration to a 70 kg person, the dosagerange would most preferably be about 35-700 mg per day.

In general, an acyclic fleximer nucleoside analogue of the presentinvention can be administered as pharmaceutical compositions by any oneof the following routes: oral, systemic (e.g., transdermal, intranasalor by suppository), or parenteral (e.g., intramuscular, intravenous orsubcutaneous) administration. Compositions can take the form of tablets,pills, capsules, semisolids, powders, sustained release formulations,solutions, suspensions, elixirs, aerosols, or any other appropriatecompositions.

The choice of formulation depends on various factors such as the mode ofdrug administration and bioavailability of the acyclic fleximernucleoside analogue. For delivery via inhalation the compound can beformulated as liquid solution, suspensions, aerosol propellants or drypowder and loaded into a suitable dispenser for administration.

A composition comprising an acyclic fleximer nucleoside analogue of thepresent invention may be combined with at least one pharmaceuticallyacceptable carrier, excipient or diluent. Some examples of acceptableexcipients are those that are non-toxic, will aid administration, and donot adversely affect the therapeutic benefit of the compound of theinvention. Such excipient may be any solid, liquid, semi-solid or, inthe case of an aerosol composition, gaseous excipient that is generallyavailable to one of skill in the art.

Solid pharmaceutical excipients useful in the invention may includestarch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk and the like. Liquid andsemisolid excipients may be selected from glycerol, propylene glycol,water, ethanol and various oils, including those of petroleum, animal,vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineraloil, sesame oil, etc. Preferred liquid carriers, particularly forinjectable solutions, include water, saline, aqueous dextrose, andglycols. Other suitable pharmaceutical excipients and their formulationsare described in Remington's Pharmaceutical Sciences, edited by E. W.Martin (Mack Publishing Company, 18th ed., 1990). The amount of anacyclic fleximer nucleoside analogue of the present invention can varywithin the full range employed by those skilled in the art. For example,a composition may contain, on a weight percent (wt %) basis, from about0.01-99.99 wt % of an acyclic fleximer nucleoside analogue of thepresent invention based on the total formulation, with the balance beingone or more suitable pharmaceutical excipients.

The pharmaceutical composition according to the invention preferablycomprises an amount of an acyclic fleximer nucleoside analogue of thepresent invention of between 5 μg and 1000 mg, preferably between 1 and500 mg, preferably between 5 and 100 mg. The ratio between the amountsby weight of an acyclic fleximer nucleoside analogue of the presentinvention and of pharmaceutically acceptable carrier is between 5/95 and95/5, preferably between 20/80 and 80/20.

The acyclic fleximer nucleoside analogues of the present invention maybe the only active ingredients, or they may be combined with otheractive ingredients. The pharmaceutical composition according to theinvention may thus also comprise at least one other pharmaceuticalactive agent, in particular at least one other medicament used for thetreatment of viral infection. In particular, the composition accordingto the invention may also comprise, or be combined with, one or moreother antivirals. Generally, any antiretroviral may be combined, namelyreverse transcriptase inhibitors, in particular nucleoside or nucleotideand non-nucleoside inhibitors, protease inhibitors, entry inhibitors,integrase inhibitors, etc.

The acyclic fleximer nucleoside analogues of the present invention orcompositions comprising same may be administered in various ways and invarious forms. Thus, they may be administered systemically, orally, byinhalation or by injection, for instance intravenously, intramuscularly,subcutaneously, transdermally, intra-arterially, etc., intravenous,intramuscular, subcutaneous and oral administration. For the injections,the acyclic fleximer nucleoside analogues of the present invention aregenerally conditioned in the form of liquid suspensions, which can beinjected by means of syringes or infusions, for example. In this regard,the acyclic fleximer nucleoside analogues of the present invention aregenerally dissolved in buffered, isotonic, physiological, saline, etc.,solutions which are compatible with pharmaceutical use and known tothose skilled in the art. Thus, the compositions may contain one or moreagents or carriers chosen from dispersants, solubilizing agents,stabilizers, preservatives, etc. Agents or carriers which can be used inliquid and/or injectable formulations are, in particular,methylcellulose, hydroxymethylcellulose, carboxymethylcellulose,polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, etc.

The acyclic fleximer nucleoside analogues of the present invention canalso be administered in the form of gels, oils, tablets, suppositories,powders, gel capsules, capsules, aerosols, etc. For this type offormulation, an agent such as cellulose, carbonates or starches isadvantageously used.

Generally, for the purpose of the present invention, solvates ofpharmaceutically acceptable solvents such as water and ethanol areequivalent to those not in forms of solvates.

The present invention relates to a method for treating a CoV viralinfection, comprising the administration, to a patient, of an effectiveamount of at least one acyclic fleximer nucleoside analogue of thepresent invention and/or of a composition containing same. The acyclicfleximer nucleoside analogue can further be prodrugs or in form ofcapable of releasing the active ingredient after in vivo metabolism.

EXAMPLES

In approaching the synthesis of the above described analogues apreviously published route was used to the acyclic sugar moiety and thenutilized a series of organometallic coupling techniques in order toconstruct the flexible base. Specifically, as shown below in Scheme, theroute to compound 8 involved coupling 2-aceteoxyethyl acetoxymethylether(4), synthesized according to published procedures, (20) todiiodoimidazole using modified Vorbruggen conditions.(20) This wasfollowed by removal of the more labile acetate protecting group andsubsequent protection with the more robust benzyl. Then, selectivedeiodination of the C5-iodo with EtMgBr yielded key intermediatecompound 8.

This product served as a key intermediate for a series of organometalliccoupling reactions. The first coupling partner selected was the boronicacid derivative of 2,4-dibenzyluracil (9). This analogue was chosen dueto the versatility of compound 10. As had been previously published (13)it was hypothesized it would be possible to obtain both theFlex-xanthosine and Flex-guanosine analogues from compound 10, as shownin Scheme 2. Preparation of the boronic acid compound 9 followedestablished procedures (21) and was used immediately without furthercharacterization. Using standard Suzuki reaction conditions in thepresence of freshly prepared Pd(PPh₃) compounds 4 and 10 were obtainedin a 45% yield. In order to obtain the xanthosine analogue, compound 10was treated with Pd/C to remove the benzyl protecting groups to yieldfinal compound 1 in a 26% yield. It was also discovered that by loweringthe temperature of the reaction, the benzyl groups could be selectivelyremoved from the base. This is convenient, as it would allow for futurefunctionalization of compound 11 without the need to selectivelyreprotect the free hydroxyl.

In an attempt to obtain the guanosine analogue, compound 10 was treatedwith methanolic ammonia under pressure, at 210 C to yield theintermediate diamino compound 12. Unfortunately, the reaction resultedin a single site transformation at the 4-position. Due to extremereaction conditions and low yield this route was abandoned. Ultimately,the guanosine analogues were obtained through Stille coupling of keyintermediate compound 8 with the appropriate pyrimidine compound 13.Pyrimidine 13 was prepared from commercially available2-amino-5-chloro-6-methoxy pyrimidine according to published procedures.The subsequent coupling was completed using modified Stille couplingconditions based on the findings of Mee et al.(22)

In order to acquire final compound 2, a selective deprotection was usedto remove the benzyl group, while leaving the 6-methoxy intact. This wasdone with Pd/C in the presence of ammonium formate in EtOH under refluxfor 18 h. This yielded compound 2 in a 24% yield, with the remainder(52%) retrievable as starting material, which could be recycled. Thefinal deprotection was accomplished with BBr₃ at room temperature toyield compound 3 in a 24% yield as shown below in Scheme 3.

The effect of numerous target fleximer analogues on the replication ofHCoV-NL63 was evaluated. HCoV-NL63 infection does not result in fullcytopathy in the Vero-118 cell culture model used. For this reason, theantiviral effect was analyzed microscopically by scoring virus-inducedcytopathogenic effects (CPE) in each well on a scale of 1 (mild) to 5(severe). These scores were then used to calculate the percentage ofinhibition by normalization to control wells.

As shown in Table 1, in contrast to acyclovir, one of the testednucleosides, nucleoside 2, demonstrated selective antiviral activity(CC50>10×).

TABLE 1 Antiviral activity of nucleoside analogues HCoV-NL63 in SARS-CoVin Vero118 MERS-CoV in Huh7 MERS-CoV in Vero VeroE6 Cmpd. EC50^(a)CC50^(b) EC50^(a) CC50^(b) EC50^(a) CC50^(b) EC50^(a) CC50^(b) 1 92 ±68 >200 ND^(c) ND^(c) ND^(c) ND^(c) ND^(c) ND^(c) 2 8.8 ± 1.5 120 ± 3713.5 ± 0.0 54.0 ± 1.7 10.1 ± 1.2 77.2 ± 50.1 28.1 ± 0.2 90.8 ± 7.13 >200 >200 ND^(c) ND^(c) ND^(c) ND^(c) ND^(c) ND^(c)Acyclovir >100 >100 >1000 >1000 >1000 >1000 >1000 >1000 17 ND^(c) ND^(c)116.2 ± 8.6   >400 71.3 ± 1.6 359.3 ± 95.4  172.1 ± 41.4  >400 19 ND^(c)ND^(c)  5.3 ± 0.7 23.4 ± 0.4  3.4 ± 0.3 17.3 ± 4.8  11.9 ± 0.2 35.0 ±7.1 ^(a)EC50: Effective concentration showing 50% inhibition ofvirus-induced CPE (in μM). ^(b)CC50: Cytotoxic concentration showing 50%inhibition of cell survival (in μM). ^(c)ND: Not determined.

Based on these results the activity of nucleoside compound 2 was alsoevaluated on the more pathogenic viruses MERS-CoV and SARS-CoV.Infection of Huh7 and Vero cells with MERS-CoV and Vero cells withSARS-CoV resulted in complete CPE. This allowed for the quantificationof the antiviral effect by using a commercial cell viability assay asdescribed previously.(23-24) Inhibition of virus-induced CPE (i.e.,enhanced cell viability compared to untreated, virus-infected cells) wasdetermined in the presence of different compound concentrations. Theresults depicted in Table 1 indicate that compound 2 can block thereplication of MERS-CoV but not SARS-CoV while acyclovir had no effect(Table 1).

The effect of compound 2 on HCoV-NL63 is significant with an EC50<10 μMand a CC50>100 μM. The results showed that compound 2 reduced theviability of different cell lines at different concentrations suggestinga cell-specific effect. The different sensitivity of the cell linestowards this nucleoside could be caused by differences in growth rate,compound uptake and metabolism, or other cell line-specificcharacteristics. Notably compound 19 can block the replication ofMERS-CoV and SARS-CoV had an EC50<12 μM and a CC50 almost three (3)times the EC50.

The present invention provides for the design, synthesis and screeningof a series of novel nucleoside analogues that employ a strategy ofcombining the flex-base motif with the flexible acyclic sugar scaffoldof the FDA-approved drug acyclovir. The results herein show that thisapproach produces medicinally relevant molecules capable of inhibitingHCoV-NL63, MERS-CoV and SARS-CoV replication in cell culture. Althoughthe parental compound, acyclovir, serves as a polymerase inhibitor,(25-26) it is yet unclear how these novel analogues disrupt viralreplication although it is theorized that such polymerase inhibitoractivity is relevant to the current compounds.

Moreover, a comparison of the activity profiles of compounds 2 and 3indicates that the methoxy group of compound 2 may be serving as aprodrug, as has been established in other antiviral nucleosideanalogues.(27)

Methods and Materials

All chemicals and reagents listed in this section were purchased throughcommercially available sources unless otherwise noted. All reactions runin CH₂Cl₂, CH₃CN, and THF were obtained from a solvent purificationsystem (SPS, Model: mBraun Labmaster 130). All reactions run inanhydrous DMF, CH₃OH and pyridine were obtained from Sigma-Aldrich orAcros Organics. All ¹H and ¹³C NMR spectra were obtained from a JEOL ECX400 MHz NMR. All ¹H NMR spectra were referenced to internaltetramethylsilane (TMS) at 0.0 ppm. The spin multiplicities areindicated by the symbols s (singlet), d (doublet), dd (doublet ofdoublets), t (triplet), q (quartet), m (multiplet), and br (broad). Allreactions were monitored by thin layer chromatography (TLC) on 0.25 mmprecoated glass plates. All flash column chromatography was run on aTeledyne Isco Combiflash Rf system. Purity of the tested compoundswas >95% based on LC, HRMS and ¹H NMR unless otherwise stated. Meltingpoints are uncorrected. All mass spectra (MS) were recorded and obtainedfrom the Johns Hopkins Mass Spectrometry Facility. The FAB mass spectrawere obtained using double focusing magnetic sector mass spectrometerequipped with a Cs ion gun and fourier transform ion cyclotron resonanceequipped with ESI source.

2-((4,5-diiodo-1H-imidazol-1-yl)methoxy)ethyl acetate (Compound 5)4,5-Diiodoimadazole (15.1 g, 47 mmol) and 2-aceteoxyethylacetoxymethylether (Compound 4, 10.0 g, 57 mmol) were dissolved inanhydrous acetonitrile (150 mL) under inert atmosphere.N,O-bis(trimethylsilyl) acetamide (70.0 mL, 284 mmol) was added. Thereaction mixture was stirred at rt for 5 h, and then cooled to 0° C.Trimethylsilyl triflouromethane sulfonate (14.0 mL, 71 mmol) was addedslowly and then the solution was heated to 90° C. and stirred for 12 h.The reaction mixture was quenched by addition of aq. NaHCO₃ (30 mL) andstirred for 30 min. The solution was separated and the aqueous layer wasextracted with CH₂Cl₂ (2×50 mL). The organic layers were combined,washed with H₂O (3×50 mL) and brine (50 mL), and dried over MgSO₄. Thesolvent was removed under reduced pressure. The resultant syrup waspurified by flash chromatography over silica gel to obtain colorlesssolid compound 5. (11.6 g, 47%). ¹H NMR (CDCl₃, 400 MHz): δ 7.73 (1H,s), 5.35 (2H, s), 4.19 (2H, t, J=4.6 Hz), 3.64 (2H, t, J=4.6 Hz), 2.06(3H, s). ¹³C NMR (CDCl₃, 100 MHz): δ 170.9, 141.8, 97.5, 81.9, 78.1,66.9, 62.8, 21.0. HRMS calcd for C₈H₁₀I₂N₂O₃ 436.8859, found 436.8860[M+H⁺].

2-((4,5-diiodo-1H-imidazol-1-yl)methoxy)ethan-1-ol (Compound 6) Compound5 (11.6 g, 27 mmol) was dissolved in ethanol (100 mL). NH₄OH was addedslowly until signs of precipitation occurred. The reaction mixturestirred at rt for 16 hr. The majority of solvent was removed via airstream to produce light yellow slurry. The solid was filtered off andwashed with ice cold H₂O (50 mL) to yield light yellow solid compound 6(7.8 g, 74%). Mp 122.8-124.1° C.; ¹H NMR (CDCl₃, 400 MHz): δ 7.74 (1H,s), 5.38 (2H, s), 3.75 (2H, t, J=4.56), 3.56 (2H, t, J=4.56). ¹³C NMR(CDCl₃, 100 MHz): δ 141.8, 97.5, 81.9, 78.2, 70.1, 61.6. HRMS calcd forC₆H₈I₂N₂O₂ 394.8753, found 394.8755 [M+H⁺].

1-((2-(benzyloxy)ethoxy)methyl)-4,5-diiodo-1H-imidazole (Compound 7)Sodium hydride (95%, 0.91 g, 38 mmol) was added to a stirred solution ofcompound 6 (10.0 g, 25 mmol) in anhydrous THF (150 mL) at 0° C. underinert atmosphere. The mixture was stirred at room temperature for 3 h.Tetrabutylammonium iodide (2.3 g, 6 mmol) and benzyl bromide (4.5 mL, 38mmol) was added. The mixture was stirred at room temperature for 12 h,followed by quenching with ethanol (20 mL). The solvent was removedunder reduced pressure; H₂O (200 mL) was added and the mixture extractedwith CH₂Cl₂ (3×100 mL). The organic extracts were combined, washed withbrine (200 mL), and dried over MgSO₄. The solvent was removed underreduced pressure to give a pale brown syrup. Flash chromatography oversilica gel gave compound 7 as a colorless syrup (8.5 g, 70%). ¹H NMR(CDCl₃, 400 MHz): δ 7.72 (1H, s), 7.31-7.34 (5H, m), 5.38 (2H, s), 4.53(2H, s), 3.61 (4H, s). ¹³C NMR (CDCl₃, 100 MHz): δ 142.0, 137.8, 128.6,127.94, 127.88, 97.3, 81.9, 78.3, 73.6, 69.4, 68.1. HRMS calcd forC₁₃H₁₄I₂N₂O₂484.9223, found 484.9225 [M+H⁺].

1-((2-(benzyloxy)ethoxy)methyl)-4-iodo-1H-imidazole (Compound 8)Compound 7 (10.0 g, 25 mmol) was dissolved in anhydrous THF (50 mL)under inert atmosphere. The reaction mixture was taken to −15° C. andEtMgBr (1.7 mL 3M in THF, 5.0 mmol) was added dropwise in two portions30 min apart. The solution was allowed to come to rt and stirred for 4hr. The reaction was quenched by addition of aq. sat. NH₄Cl (20 mL). Thesolution was separated and the aqueous layer was extracted with CH₂Cl₂(3×20 mL). The organic layers were combined, washed brine (20 mL), anddried over MgSO₄. The solvent was removed under reduced pressure. Theresultant syrup was purified by flash chromatography over silica gel toobtain yellow syrup compound 8 (7.0 g, 78%). ¹H NMR (CDCl₃, 400 MHz): δ7.48 (1H, d, J=1.36), 7.28-7.34 (5H, m), 7.13 (1H, d, J=1.36), 5.29 (2H,s), 4.52 (2H, s), 3.55-3.61 (4H, m). ¹³C NMR (CDCl₃, 100 MHz): δ 138.9,137.8, 128.6, 127.9, 127.8, 124.6, 82.9, 76.6, 73.5, 69.3, 68.1. HRMScalcd for C₁₃H₁₅IN₂O₂ 359.0256, found 359.0257 [M+H⁺].

2,4-bis(benzyloxy)-5-(1-((2-(benzyloxy)ethoxy)methyl)-1H-imidazol-4-yl)pyrimidine(Compound 10) A mixture of compound 8 (75.2 mg, 0.21 mmol) and compound9 (100 mg, 0.28 mmol) and Pd(PPh₃)₄ (33 mg, 0.028 mmol) in DME (5 mL)was stirred at r.t. under argon for 10 min. To this mixture was addedcompound 9 (103 mg, 0.03 1 mmol) in DME (5 mL). Sat. aq NaHCO₃ (10 mL)was added and the mixture refluxed under argon for 4 h. The soln wascooled to r.t. and the DME layer separated and set aside. The aqueouslayer was then extracted with EtOAc (3×25 mL), and the organic extractswere combined with the DME layer, washed with brine (50 mL), and driedover MgSO₄. The solvent was removed under reduced pressure to give apale brown syrup. Flash chromatography over silica gel gave compound 10(30 mg, 21%) as a yellow syrup. ¹H NMR (CDCl₃, 400 MHz): δ 9.07 (1H, s),7.62-7.69 (5H, m), 7.41-7.49 (5H, m), 7.27-7.38 (5H, m), 5.53 (2H, s),5.45 (2H, s), 5.31 (2H, s), 4.5 (2H, s) 3.52-3.58 (4H, m). ¹³C NMR(CDCl₃, 100 MHz): δ 166.3, 163.1, 156.2, 137.9, 137.3, 136.8, 136.3,134.5, 133.1, 132.2, 132.1, 132.04, 132.03, 128.72, 128.67, 128.54,128.50, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 118.3, 109.6, 76.8,73.4, 69.3, 69.2, 68.8, 68.0. HRMS calcd for C₃₁H₃₀N₄O₄ 523.2345, found523.2335 [M+H⁺].

5-(1-((2-hydroxyethoxy)methyl)-1H-imidazol-4-yl)pyrimidine-2,4-diol(Compound 1) A mixture of compound 10 (30 mg, 0.06 mmol), 10% Pd/C (60mg), and ammonium formate (38 mg, 0.06 mmol) in EtOH (10 mL) was heatedunder reflux for 18 h. The mixture was filtered through Celite and theCelite pad was washed several times with hot EtOH. The combined filtratewas evaporated under reduced pressure. The residue was purified by flashchromatography over silica gel to yield white solid compound 1 (9 mg,60%). ¹H NMR ((CD₃)₂SO, 400 MHz): δ 11.22 (1H, br), 10.94 (1H, br), 7.79(1H, s), 7.76 (1H, s), 7.62 (1H, s), 5.34 (2H, s), 3.42 (2H, t, J=4.6),3.36 (2H, t, J=4.6). ¹³C NMR ((CD₃)₂SO, 100 MHz): δ 162.9, 151.1, 138.0,136.4, 134.0, 117.4, 107.8, 76.2, 70.4, 60.4. HRMS calcd forC₁₀H₁₂N₄O₄253.0937, found 253.0938 [M+H⁺].

5-(1-((2-(benzyloxy)ethoxy)methyl)-1H-imidazol-4-yl)pyrimidine-2,4-diol(Compound 11) The title compound was prepared from compound 10 in thesame manner as described above altering only the temperature of thereaction to 60° C. Yield 35%, white solid. ¹H NMR ((CD₃)₂SO₄, 400 MHz):δ 11.22 (1H, br), 10.94 (1H, br), 7.79 (1H, s), 7.76 (1H, d, 0.92), 7.63(1H, d, J=0.92), 7.22-7.32 (5H, m), 5.35 (2H, s), 4.42 (2H, s), 3.51(4H, m). ¹³C NMR ((CD₃)₂SO₄, 100 MHz): δ 162.7, 150.9, 138.9, 138.0,136.4, 134.0, 128.8, 128.0, 127.9, 117.2, 107.8, 76.1, 72.5, 69.2, 68.0.HRMS calcd for C₁₇H₁₈N₄O₄343.1406, found 343.1407 [M+H⁺].

5-(1-((2-(benzyloxy)ethoxy)methyl)-1H-imidazol-4-yl)-4-methoxypyrimidin-2-amine(Compound 14) A mixture of compound 8 (233 mg, 0.65 mmol), compound 13(350 mg, 0.84 mmol), Pd(PPh₃)₄ (23 mg, 0.05 mmol), CuI, (23 mg, 0.12mmol), and tetrabutyl ammonium fluoride trihydrate (410 mg, 1.3 mmol) inanhydrous DMF (10 mL) was heated at 45° C. for 18 h. The mixture wasfiltered through Celite and diluted with EtOAc (5 mL), washed withbrine, and dried over MgSO₄. The organic solvents were removed and theproduct was purified by flash chromatography to give compound 14 (96 mg,42%) as a red oil. ¹H NMR ((CD₃)₂SO, 400 MHz): δ 8.84 (1H, s), 7.79 (1H,s), 7.40 (1H, s), 7.20-30 (5H, m), 6.56 (2H, bs) 5.37 (2H, s), 4.41 (2H,s), 3.91 (3H, s) 3.50-3.55 (4H, m). ¹³C NMR ((CD₃)₂50, 100 MHz): δ165.8, 162.3, 155.5, 138.8, 138.1, 134.8, 128.7, 128.0, 127.9, 117.2,104.7, 76.2, 72.6, 69.3, 67.9, 53.7. HRMS calcd C₁₈H₂₁N₅O₃356.1723,found 356.1729 [M+H⁺].

2-((4-(2-amino-4-methoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethan-1-ol(Compound 2) A mixture of compound 14 (94 mg, 0.27 mmol), 10% Pd/C (60mg), and ammonium formate (167 mg, 2.7 mmol) in EtOH (10 mL) was heatedunder reflux for 18 h. The mixture was filtered through Celite and theCelite pad was washed several times with hot EtOH. The combined filtratewas evaporated under reduced pressure. The residue was purified by flashchromatography over silica gel to yield pink syrup compound 2 (17 mg,22%). ¹H NMR (CD₃OD, 400 MHz): δ 8.54 (1H, s), 7.82 (1H, d, J=0.92),7.51 (1H, d, J=0.92), 5.43 (2H, s), 4.02 (3H, s), 3.63 (2H, t, J=4.56),3.51 (2H, t, J=4.56). ¹³C NMR (CD₃OD, 100 MHz): δ 166.4, 161.8, 153.8,137.4, 134.2, 117.5, 104.8, 76.5, 70.0, 60.6, 52.8. HRMS calcdC₁₁H₁₇N₅O₃ 266.1253, found 266.1247 [M+H⁺].

2-amino-5-(1-((2-hydroxyethoxy)methyl)-1H-imidazol-4-yl)pyrimidin-4(3H)-one(Compound 3) Compound 2 (9.0 mg, 0.034 mmol) was dissolved in anhydrousCH₂Cl₂ (5 mL) under inert atmosphere and brought to −78° C. BBr₃ (350μL, 0.34 mmol) was added. The reaction mixture was stirred at rt 48 hr.Solvent removed by air stream. Residue was recrystallized in EtOH toproduce white solid compound 3 (4.2 mg, 49%). ¹H NMR ((CD₃)₂SO₄, 400MHz): δ 9.19 (1H, br), 8.24 (1H, s), 8.06 (1H, s) 5.58 (2H, s),3.48-3.52 (4H, m), 2.47. ¹³C NMR ((CD₃)₂SO₄, 100 MHz): δ 159.2, 154.2,145.8, 136.2, 127.6, 118.2, 104.5, 78.4, 71.7, 60.3. HRMS calcdC₁₀H₁₃N₅O₃252.1097, found 252.1089 [M+H⁺].

2-((4-iodo-1H-imidazol-1-yl)methoxy)ethyl acetate (Compound 18)4(5)-Monoiodoimidazole (3.8 g, 19.3 mmol) and compound 4 (3.4, (4.1 g,23.2 mmol) were dissolved in anhydrous CH₃CN (150 mL) under inertatmosphere. N,O-bis(trimethylsilyl) acetamide (28.6 ml, 115.8 mmol) wasadded. The reaction mixture was stirred at rt for 5 h, and then cooledto 0° C. Trimethylsilyl triflouromethane sulfonate (5.7 mL, 25.6 mmol)was added slowly and then the solution was heated to 90° C. and stirredfor 12 h. The reaction mixture was quenched by addition of aq. NaHCO₃(30 mL) and stirred for 30 min. The solution was separated and theaqueous layer was extracted with CH₂Cl₂ (2×50 mL). The organic layerswere combined, washed with H₂O (3×50 mL) and brine (50 mL), and driedover MgSO₄. The solvent was removed under reduced pressure. Theresultant syrup was purified by flash chromatography over silica gel toobtain yellow syrup 18. (346 mg, 6%). ¹H NMR (CDCl₃, 400 MHz): δ 7.38(1H, d, J=0.92 Hz), 7.01 (1H, d, J=0.88 Hz), 5.14 (2H, s), 3.95 (2H, t,J=4.6 Hz), 3.45 (2H, t, J=4.6 Hz) 1.84 (3H, s). ¹³C NMR (CDCl₃, 100MHz): δ 170.8, 139.2, 124.6, 82.7, 76.4, 66.8, 62.8, 20.9. NMS calcd forC₈H₁₁I1N₂O₃ 310.9, found 311.0[M+H⁺].

2-((4-(2-amino-4-methoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethylacetate (Compound 19) A mixture of compound 18 (173 mg, 0.56 mmol),compound 13 (845 mg, 2.04 mmol), PdCl₂(PPh₃)₂ (143 mg, 0.2 mmol), CuI(143 mg), and TBAF.3H₂O (1.0 g, 3.14 mmol) in anhydrous DMF (25 mL) washeated at 60° C. for 18 h. The mixture was filtered The product wasdried on Celite and purified by flash chromatography to give 19 (51.6mg, 30%) as a yellow oil. ¹H NMR (CD₃OD, 400 MHz): δ 8.54 (1H, s), 7.84(1H, s), 7.51 (1H, s), 5.42 (2H, s), 4.15 (2H, t, J=4.6 Hz), 4.03 (3H,s), 3.67 (2H, t, J=4.6 Hz), 1.97 (3H, s). ¹³C NMR (CD₃OD, 100 MHz): δ171.3, 166.7, 161.7, 153.2, 137.2, 134.1, 117.6, 105.5, 76.4, 66.6,62.9, 52.7, 19.3. NMS calcd C₁₃H₁₇N₅O₄308.1, found 308.3[M+H⁺].

NL63-CoV Antiviral Assay.

This assay was performed as described previously.(28) Both the humancoronavirus NL63 strain and the Vero118 cell line were kindly providedby Ron A. Fouchier (Erasmus Medical Center, Rotterdam, The Netherlands).The HCoV-NL63 strain was isolated from an 8 months old boy sufferingfrom pneumonia.(29) A previously undescribed coronavirus associated withrespiratory disease in humans. Vero-118 cells are a subclone of Vero-WHOcells(30), and were cultured in Iscove's Modified Dulbecco's Medium(Life Technologies, Gent, Belgium—cat no 21980-032) with 10% FBS, 100 IUpenicillin/mL and 100 μg streptomycin/mL. Cells were split ¼ twiceweekly. For the antiviral assay Vero-118 cells in 96-well plate formatwere infected with HCoV-NL63 (MOI=0.01, 200 μL cell culture, 20,000cells/well, IMDM 5% FBS medium). Cultures were incubated subsequentlyfor 5 days at 35° C. and the viral cytopathic effect and cell viabilitywere scored microscopically. Scores were normalized and percentinhibition calculated.

MERS-CoV and SARS-CoV Antiviral Screening Assays.

Cell-based antiviral screening assays were performed as describedpreviously.(23-24) In brief, Huh7, Vero, and VeroE6 cells were seeded intransparent 96-well plates at a density of 10⁴ (Huh7, VeroE6) or 2×10⁴(Vero) cells per well, respectively. After overnight growth, Vero andHuh7 cells were infected with MERS-CoV (strain EMC/2012) and VeroE6cells were infected with SARS-CoV (strain Frankfurt-1) at an MOI of0.005. All work with live MERS-CoV and SARS-CoV was performed insidebiosafety cabinets in a biosafety level 3 facility at the LeidenUniversity Medical Center. Infected cells were given compound 2 or DMSO(solvent control) prior to infection. For EC₅₀ determination, two (forHuh7) or three days (for Vero and VeroE6 cells) after incubation,differences in cell viability caused by virus-induced CPE or bycompound-specific side effects were analyzed using the CellTiter 96®AQ_(ueous) Non-Radioactive Cell Proliferation Assay (Promega), accordingto the manufacturer's instructions. Absorbance (A₄₉₀) was measured usinga Berthold Mithras LB 940 96-well plate reader. For CC₅₀ determination,cytotoxic effects caused by compound treatment alone were monitored inparallel plates containing mock-infected cells. EC₅₀ and CC₅₀ valueswere calculated with GraphPad Prism 5 software using the nonlinearregression model using the results of two independent experiments.

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That which is claimed is:
 1. An acyclic fleximer nucleoside analoguehaving antiviral activity selected from the following compounds:

or a pharmaceutically acceptable salt, hydrate, prodrug or solvatethereof.
 2. A acyclic fleximer nucleoside having the followingstructure:

or a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.
 3. A composition comprising the acyclic fleximernucleoside analogue according to claim 1 and a pharmaceuticallyacceptable carrier.
 4. The composition according to claim 3, furthercomprising an additional antiviral agent.
 5. A composition comprisingthe acyclic fleximer nucleoside analogue according to claim 2, and apharmaceutically acceptable carrier.
 6. The composition according toclaim 5, further comprising an additional antiviral agent.
 7. A methodof treating and/or reducing the effects of a coronavirus in a subject inneed of such treatment, the method comprising administering atherapeutically effective amount of an acyclic fleximer nucleosideanalogue selected from the group consisting of:

or a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.
 8. A method of treating and/or reducing the effects ofa coronavirus in a subject in need of such treatment, the methodcomprising administering a therapeutically effective amount of anacyclic fleximer nucleoside analogue having the following structure:

or a pharmaceutically acceptable salthydrate, prodrug or solvatethereof.
 9. The method of claim 8, wherein the acyclic fleximernucleoside analogue is structure 19 and the coronavirus is severe acuterespiratory syndrome (SARS) or Middle East respiratory syndrome (MERS).10. The method of claim 8, wherein the acyclic fleximer nucleosideanalogue is in a composition further comprising a pharmaceuticallyacceptable carrier.
 11. The method of claim 8, wherein the acyclicfleximer nucleoside analogue is in a composition further comprising anadditional antiviral agent.
 12. The method of claim 8, wherein thetherapeutically effective amount of the acyclic fleximer nucleosideanalogue is from 0.05 to 50 mg per kilogram body weight of the subjectper day.